Annual Report HS02243-01 Since food intake and energy metabolism is synchronized to the circadian cycle, we investigated the role of AMPK in generating circadian rhythm. One of the regulators of the period length is casein kinase I&#949;(CKI&#949;), which by phosphorylating and inducing the degradation of the circadian clock component mPer2, shortens the period length. AMPK phosphorylates Ser 389 of CKI&#949;, resulting in increased CKIe activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK &#945;2 knockout mice. In order to further characterize the role of AMPK in circadian rhythm, we characterized the circadian rhythm in different tissues of AMPK &#945;1 and &#945;2 knockout mice. We found that the two isoforms of AMPK regulate circadian rhythm patterns in a tissue -dependent manner. Conversely, disruption of circadian rhythm may affect energy metabolism. To test this, we studied the metabolic phenotype of mPer2-deficient mice. We found that production of anorexic neuropeptide &#945;MSH during the inactive period is diminished in the hypothalamus of mPer2-deficient mice, leading to overeating and fat accumulation. Restoring &#945;MSH levels via peripheral injection prevented overeating and fat accumulation in mPer2-deficient mice but not in wild-type mice. Sirt1, which lies downstream of AMPK, may play an important role in aging-related diseases, but the mechanisms by which it is regulated is poorly understood. In response to DNA damage, casein kinase 2 (CK2)-mediated phosphorylation of Ser 154, 649, 651 and 683 increase not only its deacetylase activity but its affinity for substrates as well. Two of the CK2 phosphorylations sites lie within a 25 a.a. region in the C-terminal domain that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an on switch for the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Sirt1 activity is also partially suppressed by ATP in a concentration-dependent manner. ATP binds to the ESA region and inactivates the ESA on switch. Abdominal fat is associated with a variety of health risks, but subcutaneous fat has protective effects. Aging is not only associated with increased abdominal fat depot but loss of subcutaneous fat depot. Increasing adipocytes in the subcutaneous fat depot may reduce fat accumulation in the abdominal depot. Using in vitro adipogenesis models, we discovered that PML, the expression of which decreased during adipogenesis, is a suppressor of adipogenesis and PML-deficiency enhanced adipogenesis. Loss of PML increased expression of the adipogenic transcription factors, CCAAT/enhancer binding protein &#945;(C/EBP&#945;) and peroxisome proliferator-activated receptor &#947;(PPAR&#947;). PML-deficient mice gained body weight faster and had more fat mass in diet-induced obesity model despite having normal activity levels and metabolic rate. However, they were not insulin-resistant or glucose-intolerant and did not develop obesity-associated inflammation. These findings indicate that PML-deficiency may provide insights into therapeutic strategies for increasing adipogenesis without causing metabolic disease.